RESUMEN
Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise.
Asunto(s)
Dodecil Sulfato de Sodio/análisis , Espectrofotometría/métodos , Concentración de Iones de Hidrógeno , Luz , Límite de Detección , Reproducibilidad de los Resultados , Solventes/químicaRESUMEN
Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales , Immunoblotting/métodos , Preparaciones Farmacéuticas/análisis , Acridinas/química , Antibacterianos/química , Antibacterianos/inmunología , Antiinflamatorios/química , Antiinflamatorios/inmunología , Anticuerpos Monoclonales/química , Anticonvulsivantes/química , Anticonvulsivantes/inmunología , Carbamazepina/química , Carbamazepina/inmunología , Colodión/química , Ciclosporina/química , Ciclosporina/inmunología , Gentamicinas/química , Gentamicinas/inmunología , Hidrocortisona/química , Hidrocortisona/inmunología , Inmunosupresores/química , Inmunosupresores/inmunología , Metotrexato/química , Metotrexato/inmunología , Reproducibilidad de los Resultados , Sirolimus/química , Sirolimus/inmunología , Tacrolimus/química , Tacrolimus/inmunologíaRESUMEN
OBJECTIVE: We investigated the mechanism by which the ARCHITECT cyclosporine (CsA) chemiluminescent microparticle immunoassay (CMIA) eliminates cross-reactivity to CsA metabolites AM1 and AM9, despite its use of a monoclonal antibody which shows cross-reactivity in fluorescence polarization immunoassays. DESIGN AND METHODS: The CMIA was accomplished by incubating an extracted blood sample with magnetic microparticles coated with a very low amount of anti-CsA antibody. After a wash step the microparticles were incubated with a chemiluminescent CsA tracer, followed by a second wash step and measurement of chemiluminescence. The reagent concentrations of salt and detergent were optimized to maximize CsA binding and minimize metabolite interference. RESULTS: Elimination of CsA metabolite cross-reactivity was shown using purified metabolites and blood samples containing native CsA metabolites. The CMIA demonstrated precision and sensitivity acceptable for use in a clinical setting. CONCLUSION: We conclude that it is possible to eliminate CsA metabolite immuno-cross-reactivity by careful assay design.